The replicative history and replicative potential of human naive and memory T cells, critical parameters of lymphocyte biology, were analyzed. Telomeres are unique terminal chromosomal structures which shorten with cell division in vitro and with increased age in vivo for human somatic cells. We found that telomeres were longer in naive T cells than in memory cells from the same donors over a wide range of donor age,suggesting that differentiation of memory cells from naive precursors occurs with substantial clonal expansion. The in vitro replicative capacity of naive cells was greater than that of memory cells from the same donors. Human CD4+ naive and memory cells thus differ in in vivo replicative history as reflected in telomeric length as well as in their residual replicative capacity. Analysis of telomere length regulation in human B cells demonstrated that germinal center(GC)B cells have significantly longer telomeres than the naive B cells that are their precursors or the memory B cells that are their progeny. These results suggest the novel possibility that normal somatic cells of the B lymphocyte lineage express a mechanism capable of extending telomere length. Such a mechanism might function to extend the capacity for clonal expansion of memory and effector B cells.Telomerase, a ribonucleoprotein enzyme that is capable of synthesizing telomeric repeats, is expressed in germline and malignant cells and is absent in most normal human somatic cells. The selective expression of telomerase has thus been proposed to be a basis for the immortality of the germline and of malignant cells. When telomerase activity was analyzed in normal human T lymphocytes, it was found that telomerase is expressed at a high level in thymocytes, at an intermediate level in tonsil T cells, and at a low to undetectable level in peripheral blood T cells. Moreover, telomerase activity was highly inducible in peripheral T lymphocytes by activation through CD3 and CD28. Telomerase may thus play a role in T cell development and in the capacity of lymphoid cells for clonal expansion. In human tonsil B cells, telomerase is expressed at a high level in GC B cells and may provide a mechanism for the telomere lengthening that occurs in differentiation from precursor to GC B cells.Expression of the two genes encoding the necessary and sufficient components of telomerase, RNA template (TR) and reverse transcriptase catalytic component (TERT), has been analyzed, and both mRNAs have been found to be regulated during lymphocyte development and activation. We further demonstrated that telomerase enzymatic activity in vivo is determined by factors in addition to steady state RNA levels of TR and TERT. Specifically, events including phosphorylation of hTERT and translocation of TERT from cytoplasm to nucleus occur concurrent with the induction of telomerase activity in activated T cells. To further study the regulation of telomerase at a transcriptional level, a series of genetically engineered mice have been constructed including: mTERT cDNA transgenics, GFP knock-in as a reporter for mTERT transcriptional activity (also resulting in mTERT knock-out inactivation), and an hTERT BAC transgenic that allows study of human TERT regulation in human versus mouse cellular environments. In addition, the functions of telomere-associated proteins TIN-2, tankyrase-2, and tankyrase-1 are being analyzed through construction of both constitutive and conditional knockouts for each of the corresponding mouse genes. Initial studies demonstrated that constitutive inactivation of TIN-2 results in early embryonic lethality. Conditional knockouts will be used to analyze the mechanism of these effects. Tankyrase-2 knockout mice are viable and are currently being phenotyped. In addition, tankyrase-1 deficient mice have been constructed, and are being bred to homozygosity and in crosses to tankyrase-2 to screen for redundant and non-redundant functions between these two relate